X-ray contrast agent

ABSTRACT

The present invention refers to a phospholipid-based compound, that is a phospholipid to which an X-ray contrast-giving moiety has been covalently linked, liposomes comprising said compound as well as the use of said liposomes as a diagnostic or contrast agent.

TECHNICAL FIELD

The present invention is related to a phospholipid-based compoundcomprising an X-ray contrast-giving moiety, which compound in the formof liposomes in an aqueous formulation after intravenous administrationcan be used for X-ray examination of the reticuloendothelial system(RES), especially the liver.

BACKGROUND

There is in medicine a great need for a non-toxic contrast agent for theliver. In the liver, as well as in the spleen which are part of thereticuloendothelial system, there are so called Kupffer cells, thenormal task of which is to clear the blood of foreign particles. It is awell known fact that colloidal particles, such as liposomes, wheninjected into the body, are concentrated in said Kupffer cells. In anumber of diseases, such as cancer, there are, however, no such cells inthe disordered tissue. By using a particulate contrast agent, which willonly be taken up by the healthy tissue, there is therefore a possibilityto differentiate between disordered and healthy tissue.

In order to replace Thorotrast, which was introduced in 1929 andabandoned due to the development of histological changes, many attempshave been made to find a contrast agent for the liver not being toxic.All these experiments have in common that the results often werediagnostically satisfactory but nevertheless discouraging as all effortslead to unavoidable toxicity. A number of particulate contrast agentshas been developed and tested which have been based on polymericmaterials or on lipids combined with a contrast giving substance. Saidcontrast agents have been of different types such as iodinated compoundsfor use in X-ray diagnostics, conventional as well as for computedtomography (CT), magnetic materials for use in magnetic resonanceimaging (MRI) and radioactive isotopes.

For toxicological reasons, that is for avoiding the risk of radiation aswell as of introducing foreign substances, it should be of advantage touse a conventional X-ray contrast agent of the type iodinated carboxylicacid which is well known and highly compatible with the human body.

As there are only about 1% Kupffer cells in the liver, thecontrast-giving moiety of the contrast agent must not be too small; ithas been concluded that in general the contrast-giving moiety should notbe less than about 25% w/w for an uptake to be diagnosticallydetectable. In addition the size of the particles should be within theinterval 0.05-5 μm as larger particles to a great extent are caught inthe lungs and smaller particles will not be taken up by the Kupffercells.

Another problem with a particulate contrast agent to be used forparenteral administration, and intravenous administration in particular,is the demand for sterilization. When a contrast agent is to be preparedon a large scale for a medical application, one of the most importantsteps in the manufacturing process is the sterilization. In order toremove small viruses and pathogenetic materials heat sterilization isstill the only reliable method. The particle structure of many of thepreviously known particulate carriers for contrast agents, such asliposomes and starch-based particles, can generally not withstand thistreatment.

PRIOR ART

Particulate contrast agents based on lipids can be divided into two maincategories. On the one hand an appropriate contrast agent can beincluded in a carrier particle, for instance a liposome, and on theother hand the contrast agent can be linked to a lipid as such, andsubsequently by means of excipients be formed to particles, such asliposomes or emulsion droplets. In this context liposome refers to aspherical particle comprising two or more bilayers of mainlyphospholipids, in the interspace of which hydrophilic substances, forinstance water, can be included. Emulsion refers to drops of lipid,mainly consisting of triglycerides which have been stabilised preferablywith phospholipids, in an aqueous continuous phase.

Different methods have been tested in the preparation of lipid systemsbased on emulsions. Lipoidal® UF (Laboratoire Guerbert,Aulnay-sous-Bois, France) is an iodinated ethyl ester of fatty acids,which has been emulsified and used as a particulate system.

EP-A1-294 534 refers to an X-ray contrast agent emulsion for parenteral,especially intravenous administration, containing iodinated lipids, suchas triglycerides or alkyl esters of fatty acids, emulsified in anaqueous phase to which has been added a stability increasing agent andoptionally an oil or a fat. This emulsion is based on the Intralipid®concept; lipid emulsions which have been used in clinic as nutritionsolutions for many years. The emulsion mainly consists of triglyceridesfrom soybean oil and phospholipids from eggs, which have been emulsifiedto a stable emulsion having a very low toxicity.

Iodinated alkyl compounds have, however, under certain conditions provedto function as alkylating agents in vivo and also to bring aboutmacrophage activation. This is not surprising as it could be expectedthat iodinated acyl chains in the cell membrane should lead to changesaffecting general membrane functions such as fluidity etc., which inturn have regulating functions. It could therefore be concluded thatiodinated lipids should not contain iodine in the hydrocarbon chain.

DE 29 35 195 refers to an X-ray contrast agent for the liver and spleen,which consists of an iodine containing X-ray contrast giving compound ofthe type 2,4,6-triiodobenzoic acid derivative enclosed in sphericalphospholipids, that is liposomes having a multilayer structure.

U.S. Pat. No. 4,192,859 also refers to X-ray contrast media comprisingan X-ray contrast agent and a liposome as a carrier therefor. Theliposome comprises lecithin and a sterol and is said to include cavitiescontaining the contrast agent therein, i.e. the contrast agent is notchemically bonded to the liposome.

WO 88/09165 refers to an injectable aqueous composition, developed foropacifying organs for X-ray examination, which comprises at least oneiodinated organic compound opaque to X-rays encapsulated in liposomicvesicles. It is stated that the particle size of the liposome vesiclesshould be 0.15-3 μm and the ratio encapsulated iodine to lipids in theliposomic vesicles from 1.5 to 6 g/g.

A general disadvantage with the references cited above referring toliposomes as carriers for contrast agents is that the substancesentrapped by the bilayer membranes leak in tissue fluids. This bilayermembrane leakiness also prevents the liposome carriers from being heatsterilizable. There is consequently a need for contrast giving moietiesthat remain associated to the carrier particle, for instance a liposome,in the body.

Another difficulty with the liposome systems cited above is that thetechniques for producing the final dispersion often are complicated andinvolves stability problems.

DISCLOSURE OF THE INVENTION

The object of the invention is to provide a non-toxic X-ray contrastagent for the reticuloendothelial system which after intravenousadministration gives a satisfactory visualization of the liver, and inaddition is sufficiently stable to be easy to manufacture and sterilize.

The phospholipid-based compound

The present invention refers to a new phospholipid-based compound, to beprecise a phospholipid to which an X-ray contrast-giving moiety has beencovalently linked.

The phospholipid based compound of the invention can be represented bythe following formula ##STR1## wherein R' is the lipid part of thephospholipid and R" is the contrast-giving moiety of the compound, whichhas been associated to the phospholipid by a covalent coupling,preferably by an amide or ester bond.

The phospholipids according to the invention are composed of polar andnon-polar groups on a backbone molecule, generally glycerol. Also otherbackbone molecules can be used, for instance the natural sphingosinebases and sterols. Similar structures can also be synthesized fromnatural or synthetic alcohols and amines. According to a preferredaspect of the invention the phospholipids should be of natural origin,preferably membrane phospholipids.

Glycerophospholipids, that is phospholipids having a glycerol backbone,can be represented by the formula II ##STR2## wherein R₁ and R₂independently represent R₄ or OCR₄, wherein R₄ represents a saturated orunsaturated, branched or straight chain alkyl or alkylene group having7-23 carbon atoms, preferably 11-19 carbon atoms; and R₃ represents anamide or ester bonding group.

R₁ and R₂ generally are fatty acid residues of variable length. Aspreferred examples of fatty acids can be mentioned naturally occuringfatty acids, such as the saturated acids palmitic (C₁₅ H₃₁ CO) andstearic (C₁₇ H₃₅ CO) acid; the monosaturated acid oleic acid (C₁₇ H₃₃CO); and the polyunsaturated acids linoleic acid (C₁₇ H₃₁ CO) andlinolenic acid (C₁₇ H₂₉ CO).

When a phospholipid of natural origin is chosen the acyl chains will bevarying with respect to length and number of double bonds in contrast toa phospholipid of synthetic origin, in which case it will be possible toprepare the phospholipid with specific acyl chains. As an example ofthis can be mentioned that for instance phosphatidylethanolamine (PE)from soybean and egg respectively, have the fatty acid pattern accordingto Table 1, whereas a synthetic PE can consist of up to 100% C 16:0.

                  TABLE 1                                                         ______________________________________                                                       % by weight                                                    Fatty Acid       soybean PE                                                                              egg PE                                             ______________________________________                                        16:0 palmitate   21        15                                                 18:0 stearate    1         28                                                 18:1 (n-9) oleate                                                                              7         20                                                 18:1 (n-7)       1         1                                                  18:2 linoleate   63        9                                                  18:3 linolenate  6         --                                                 20:4             --        10                                                 20:6             --        13                                                 miscellaneous    1         5                                                  ______________________________________                                    

R₃ in the formula II can be represented by C₁ -C₅ alkylene, which may besubstituted or not, having a terminal NH₂ or OH group, preferably a C₁-C₂ alkylene amino group, optionally substituted by a hydroxymethyl orcarboxymethyl group. As examples can be mentioned the ethanolamine orserine residues, which both have terminal NH₂ groups suitable forcoupling of the contrast-giving substituent. Said residues are bothimportant consituents of cell membranes in mammals. Examples of othergroups are residues from other amino acids; for instance tyrosine andthreonine having terminal OH groups in the structure in the same way asserine, which can be associated with the phosphate group. For the manskilled in the art it will be easy to amend this type of group inaccordance with the invention.

Sphingophospholipids, such as sphingomyeline analogues, may contain twoamino groups and can form two different amides. Sphingophospholipids canbe based on different bases, such as sphingosine, sphingenine,sphinganine and hydroxysphinganine.

A preferred class of phospholipids is derived from natural lipids, suchas the membrane phospholipids. As examples of glycerophospholipids canbe mentioned phosphatidylethanolamine, and phosphatidylserine, as wellas phosphatidylinositol and phosphatidylglycerol. The naturallyoccurring compounds in the class normally have fatty acid residues inthe non-polar part of the molecule and are described in the literature(Schmid et al, Progress in Lipid Research 29, 1990, 1). Natural N-acylphosphatidylethanolamine can e.g. be found in microorganisms, plants(especially cereals), fish and mammals, and thus also in food.

The contrast giving moiety of the phospholipid based compound accordingto the invention is derived from a conventional iodinated X-raycontrast-giving compound, that is an iodinated carboxylic acid,preferably comprising a triiodo-phenyl group, such as a triiodinatedbenzoic acid or triiodophenyl propionic acid. Additional iodinatedcontrast-giving compounds can be found in e.g. Hoey et al, Handb. Exp.Pharmacol, vol 73 (Radiocontrast Agents), 1984, pp 23-125.

The new compounds of the invention of the formula I can be prepared bymethods known in the art for chemical coupling of an acid to an amine-or hydroxyterminated compound. Amidation of phosphatidylethanolamine isfor instance described by Dawson et al, Biochem. J. 114, 1969, 265.

Preferred phospholipid compounds of the formula I are those swelling inwater, a characteristic necessary for the ability to form liposomesspontaneously. For a phospholipid to form a liposome in excess of waterit is necessary that a lamellar liquid crystalline phase is formed, aswith phosphatidylcholine (PC). Phosphatidylethanolamine (PE) on theother hand normally favours the reversed hexagonal phase.

The compounds of the formula III ##STR3## wherein R₄ and R₄, are definedas above, and X and Y are hydrogen or substituent groups, constitute apreferred class of phospholipid-based compounds of the invention.

Also other associations between the phospholipid and the contrast-givingpart of the molecule can easily in a similar way be constructed by theman skilled in the art in order to make combinations which, when brokendown in the body, give substances previously known, preferably ofnatural origin. Such an example is acylornithines and other ornithinelipids.

The phospholipid-based liposomes

The invention also refers to phospholipid-based liposomes, that ismultilamellar vesicles, comprising a phospholipid based compound of theformula I above, that is a phospholipid to which an X-raycontrast-giving moiety has been covalently linked.

The liposomes generally are of the size 0.05-10 μm, preferably 0.1-1 μm.

The formation of the liposomes of the compound according to theinvention is facilitated by the compound swelling spontaneously in waterforming a lamellar liquid crystalline phase having a maximum watercontent of about 35% by weight. Depending on the compound used and theother conditions a spontaneous formation of liposomes can be obtainedwhen water is added to this lamellar phase. If not, the formation ofliposomes can be accomplished by mechanical dispersion of the lamellarliquid-crystalline phase in excess water, whereby the lipid bilayersform closed spherical aggregates, that is liposomes. A preferred methodfor the dispersion is stirring, for instance by means of an ultraturrax,but shaking, vortexing and rolling can also be performed. If liposomesof a smaller size are aimed at the dispersion could preferably beultrasonicated.

It should be noticed that the preparation of the liposomes according tothe invention does not require any treatment with organic solvents, suchas chloroform or dichloromethane, which are used in conventionalmethods.

If the phospholipid-based compound according to the invention does notswell spontaneously in water, it is possible to obtain liposomes by theaddition of a more polar, swellable lipid, preferablyphosphatidylcholine.

The liposome formation can be performed at room temperature or any othertemperature above 0° C. if the phase transition temperature of the acylchains (chain melting; gel-to-liquid crystals) is below the freezingpoint of water, which is the case for natural phospholipids.

To attain a more homogeneous size distribution of the liposomes, theliposomal dispersion can be extruded through a membrane filter.

In a preferred aspect of the invention liposomes can be prepared bydirect swelling of the phospholipid-based compound in an aqueous mediumwithout adding any other substances such as stabilizers etc. which arenormally required.

A phospholipid-based compound of the formula I, wherein R' is the lipidpart of a phosphatidylethanolamine and R" is a derivative of atriiodobenzoyl group, has surprisingly turned out to be able to formliposomes spontaneously in an aqueous medium. This reflects the balancedcharacter of the iodine-containing lipid with respect tohydrophilicitylipophilicity. It could be noticed that "normal" bilayerforming lipids, as for example phophatidylcholine, require mechanicalenergy in order to be dispersed into liposomes.

A preferred aqueous medium is an isotonic medium, for instance 2.6% w/wglycerol in water, 5% w/w glucose or 10% w/w sucrose. Saline, phosphatebuffered saline or any other electrolyte solution should be avoided asdispersion media, as these give rise to the formation of reversedhexagonal liquid crystals.

X-ray contrast agent

The phospholipid-based liposomes of the invention can be used as anX-ray contrast agent. A liposomal dispersion of a concentration of0.25-20% w/w is non-toxic and sufficiently stable to be heat sterilized.Liposomes having an iodine content of about 32% can be made, which makesthem suitable for X-ray diagnostics. By intravenous injection in rabbit,followed by X-ray computed tomography of the liver, a good contrasteffect was attained.

A suitable concentration of the liposomal dispersion is 10-20%. As anormal dose of iodine in an liver examination is about 5-15 g/subject,the total dose to be administered will be 80-470 ml.

When the X-ray contrast agent according to the invention is metabolizedin vive a water-soluble iodinated substance is formed by enzymaticreaction. This compound is of the same type as the X-ray contrast agentsused today of the type triiodobenzoyl derivatives. It is an importantaspect of the invention that the phospholipid-based liposomes have beenconstructed in such a way that, when broken down in the body, previouslyknown substances, preferably of a natural origin, are formed. In generalthey are decomposed by the action of phospholipases, wherebyphospholipase D in particular releases the N-acylethanolamine compoundfrom the phosphate group. The released N-acylethanolamine is metabolizedby amidases, which are of frequent occurrence e.g. in the liver ofmammals.

DESCRIPTION OF THE DRAWING

The FIGURE shows a microphoto pattern in polarized light of 5% w/wiodine-containing phosphatidylethanolamine liposomes according toExample 5 in a magnification of ×100. The spherical patterns with theMalthesian crosses are characteristic features for liposomes.

EXAMPLES

The invention will be further illustrated by the following examples.Example 1 refers to the synthesis of a phospholipid-based compound ofthe invention, Examples 2-3 to the preparation of liposomes from saidcompound and Examples 4-6 to physicochemical characterization of saidliposomes.

The dispersions of liposomes were heat sterilized and characterized asfollows:

Heat sterilization: 2-5 ml of the dispersion was transferred to a glassvial which was sealed with a rubber stopper and an aluminum cap. Thedispersion was then heat sterilized at 121° C. for 20 minutes.

Visual appearance: After heat sterilization the liposomal dispersionswere characterized visually with respect to aggregation, sedimentationand colour.

Photomicrography: An Olympus CH-2 polarizing microscope, attached to anautomatic exposure photomicrographic system with a large format Polaroidcamera back, was used. A small amount of the liposomal dispersion wastransferred onto a glass slide and covered with a slide cover. Theappearance of the sample was then observed between crossed polarizers.

Turbidity measurements: The turbidity, that is the absorbance, beforeand after heat sterilization was recorded at 600 nm on a Hitachi U-1100spectrophotometer at ambient temperature. 1 cm cuvette cells were usedand no stirring of the dispersion was performed.

Particle size measurements: The mean particle sizes of liposomes beforeand after heat sterilization were determined by dynamic light scatteringmeasurements using a Zetasizer 4 (Malvern Instruments, England) equippedwith an AZ 110 cell. The scattering angle was 90° and the temperaturewas 25° C. Data are expressed as cumulant z averages.

EXAMPLE 1

N-(2,3,5-triiodobenzoyl)-phosphatidylethanolamine ##STR4## wherein R₁and R₂ are 80% C₁₇ H₃₁ and 20% C₁₅ H₃₁.

Synthesis: 2.5 g 2,3,5-triiodobenzoic acid is refluxed with 5 ml thionylchloride for 45 minutes. The excess of thionyl chloride is distilled andthe remainder is recrystallized in CCl₄. The yield was 2.3 g having amelting point of 90°-91° C.

0.5 g PE, phosphatidylethanolamine from soybeans (Karlshamns LipidteknikAB) was dissolved in 200 ml chloroform, 20 ml triethylamine was thenadded and 0.8 g redistilled 2,3,5-triiodobenzoyl chloride. The reactionmixture was stirred for 24 hours at ambient temperature and the reactionwas followed by thin-layer chromatography until completion. The reactionmixture was washed carefully with a saturated solution of sodiumbicarbonate. The chloroform phase was dried over MgSO₄ and wasevaporated. The resulting product was recrystallized in hexane. Theyield of the final product was 75% with a purity higher than 99.5%(determined by HPLC). The structure of the final compound was confirmedby ¹³ C-NMR (CDCl₃, TMS, 101 MHz); 173.33 and 172.93 (CαO esters),168.29 (C═0 amid); 151.12 (C₁ in aromatic ring), 147.3 (C₅ in aromaticring), 135.40 (C₆ in aromatic ring), 130.27/130.04/128.17/127.98(unsaturation in aliphatic chain), 112.10 (C₄ in aromatic ring); 106.55(C₃ in aromatic ring); 93.93 (C₂ in aromatic ring); 70.53 (CHOglycerol); 64.02 (CH₂ OP glycerol); 62.68 (CH₂ O glycerol); 45.86(POCH₂); 41.7 (CH₂ N); 34.34-14.01 (aliphatic chain).

EXAMPLE 2

150 g of the compound of Example 1 was mixed with 850 g 2.6% w/wglycerol in distilled water. The mixture was equilibrated for two hourswithout stirring in order to swell the lipid. The swollen lipid was thenstirred to homogeneity and sterilized by heating at 95° C. for fiveminutes, followed by aseptic packaging in glass ampoules.

EXAMPLE 3

100 g of the compound of Example 1 was mixed with 900 g distilled waterto which 2.6% w/w glycerol had been added in order to achieve anisotonic solution. The lipid was allowed to swell in the aqueous phasefor two hours. The mixture was homogenized by ultrasonication. 5 ml ofthe resulting dispersion was sealed in glass ampoules and heatsterilized in boiling water for five minutes.

EXAMPLE 4

70 mg of the compound of Example 1 was mixed with 6.93 gmembrane-filtered water. The mixture was allowed to swell for 2 h duringslow agitation which resulted in a homogeneous dispersion.

One part of the dispersion was filtered through a 0.45 μmmembrane-filter (A). Another part of the dispersion was filtratedthrough a 0.45 μm membrane filter, followed by heat sterilization (B).The last part was untreated (C).

The turbidity was then measured and the following results were obtained:

    ______________________________________                                        Sample    Turbidity/Absorbance units                                          ______________________________________                                        A         0.657                                                               B         0.652                                                               C         0.780                                                               ______________________________________                                    

The cuvette cells were left to stand at ambient temperature for 1 month.No appreciable sedimentation could be observed after visual examination.

EXAMPLE 5

3.03 g of the compound of Example 1 was mixed with 11.79 gmembrane-filtered water och 0.38 g 99.5% glycerol. The mixture wasallowed to swell overnight during slow agitation. A part of theresulting homogeneous dispersion was diluted with 2.50% w/w glycerol inwater to give a final lipid concentration of 5.00% w/w.

EXAMPLE 6

The dispersion of Example 5 was diluted with 2.6% w/w glycerol in waterto give a concentration of 0.25% w/w and was filtered through a 0.45 μmpolycarbonate membrane filter in order to remove dust particles. Onepart of the dispersion was heat sterilized (A) whereas another part wasuntreated (8). The size distributions of the samples were then measured.

    ______________________________________                                        Sample     Average particle size, nm                                          ______________________________________                                        A          184                                                                B          199                                                                ______________________________________                                    

The results clearly show that the liposomes according to the inventionwere more or less unaffected by the heat sterilization, i.e. theliposomal structure was maintained without aggregation and subsequentsedimentation. In general the size of the particles was somewhat reducedafter the heat treatment. Furthermore, the fraction of particles largerthan 1000 nm was reduced from being ca. 5% of the total particles toless than 0.1% after heat sterilization.

Biological test

In order to evaluate the efficiency of the new iodine-containingphospholipid based liposomes as a liver-specific contrast medium atX-ray computed tomography, studies were performed in rabbits. Scans wereobtained immediately prior to and 30 minutes after intraveneousinjection of the dispersion (containing 50 mg iodine per ml solution).The concentration in the rabbit was approximately 50 mg iodine per kgbody weight. The injection rate was 20 ml per minute. The averageincrease in attenuation was 34.5 Hounsfield units (HU), which is 3 timeshigher than what was found by Ivancek when Intraiodole was used inapproximately the same concentration, and in level with what wasachieved with Intraiodole containing 300% more of iodine (ActaRadiologica 30, 1989, 409).

                  TABLE 2                                                         ______________________________________                                        Attenuation in rabbit, in HU                                                                0       30 min                                                  ______________________________________                                        Region of interest                                                                            55.3      92.2                                                (circular)      47.1      86.2                                                                56.1      89.9                                                                58.2      79.7                                                                45.4      77.6                                                                43.0      82.1                                                                42.6      86.0                                                                50.6      82.8                                                                46.6      80.9                                                                47.1      82.8                                                                          79.9                                                                          84.7                                                Mean value ± 49,2 ± 1,76                                                                          83,7 ± 1,24                                      standard deviation                                                            ______________________________________                                    

The above data show that there is an increase of the attenuation of 34.5HU 30 minutes after the administration, which is a satisfactory increasefor assessing liver tumors.

We claim:
 1. A phospholipid-based compound which comprises aphospholipid to which an x-ray contrast-giving moiety has beencovalently linked by means of an ester or an amide bond.
 2. The compoundaccording to claim 1 in which said phospholipid comprises a terminalamino group, to which the x-ray contrast-giving moiety has been linkedby means of an amide bond.
 3. The compound according to claim 1 in whichsaid phospholipid is a glycerophospholipid.
 4. The compound according toclaim 3 wherein said glycerophospholipid is selected from the groupconsisting of phosphotidylethanolamine, phosphotidylserine,phosphotidylinositol and phosphotidyglycerol.
 5. The compound accordingto claim 1 in which said x-ray contrast-giving moiety is an iodinatedcarboxylic acid.
 6. The compound according to claim 5 in which saidiodinated carboxylic acid is selected from the group consisting oftriiodinated benzoic acid and triiodophenyl propionic acid.
 7. Acompound according to claim 1 wherein said phospholipid-based compoundswells spontaneously in an isotonic aqueous solution. 8.Phospholipid-based liposomes comprising the phospholipid-based compoundof claim
 1. 9. The phospholipid-based liposomes of claim 8 which aredispersed in an aqueous environment.
 10. Liposomes as claimed in claim 8which are formed without addition of excipients.
 11. Liposomes asclaimed in claim 8 which are of the size 0.05-10 μm.
 12. Liposomes asclaimed in claim 11 which are of the size 0.1-1 μm.
 13. A method ofpreparing an x-ray contrast agent which comprises covalently linking anx-ray contrast-giving moiety to a phospholipid by means of an ester oran amide bond.
 14. The method of claim 13 wherein said phospholipidcomprises a terminal amino group, to which the x-ray contrast-givingmoiety has been linked by means of an amide bond.
 15. The method ofclaim 13 wherein said phospholipid is a glycerophospholipid.
 16. Themethod of claim 15 wherein said glycerophospholipid is selected from thegroup consisting of phosphotidylethanolamine, phosphotidylserine,phosphotidylinositol and phosphotidylglycerol.
 17. The method of claim13 wherein said x-ray contrast-giving moiety is an iodinated carboxylicacid.
 18. The method of claim 17 wherein said iodinated carboxylic acidis selected from the group consisting of triiodinated benzoic acid andtriiodophenyl propionic acid.
 19. The method of claim 13 wherein saidphospholipid-based compound swells spontaneously in an isotonic aqueoussolution.
 20. Method of providing a non-toxic x-ray contrast agent forx-ray diagnosis comprising:administering to a host a phospholipid-basedcompound comprising a phospholipid to which the x-ray contrast givingmoiety has been linked by means of an amide bond to a terminal aminogroup of said phospholipid.
 21. The method of claim 20 wherein saidphospholipid is a glycerophospholipid.
 22. The method of claim 21wherein said glycerophospholipid is selected from the group consistingof phosphotidylethanolamine, phosphotidylserine, phosphotidylinositoland pholsphtidylglycerol.
 23. The method of claim 20 wherein said x-raycontrast-giving moiety covalently linked to said phospholipid is aniodinated carboxylic acid.
 24. The method of claim 23 wherein saidiodinated carboxylic acid is selected from the group consisting oftriiodinated benzoic acid and triiodophenylpropionic acid.
 25. Themethod of claim 20 wherein said phospholipid-based compound swellsspontaneously in an isotonic aqueous solution.
 26. The method of claim13 further comprising forming phospholipid-based liposomes whichcomprise the compound resulting from said linking.
 27. The method ofclaim 26 wherein said liposomes are dispersed in an aqueous environment.28. The method of claim 26 wherein said liposomes are formed withoutaddition of excipients.
 29. The method of claim 26 wherein saidliposomes are of the size 0.05-10 μm.
 30. The method of claim 29 whereinsaid liposomes are of the size 0.1-1 μm.
 31. The method of claim 20wherein said diagnosis comprises visualizing the reticuloendothelialsystem.